Hyperpolarized 15N-pyridine Derivatives as pH-Sensitive MRI Agents

Highly sensitive MR imaging agents that can accurately and rapidly monitor changes in pH would have diagnostic and prognostic value for many diseases. Here, we report an investigation of hyperpolarized 15N-pyridine derivatives as ultrasensitive pH-sensitive imaging probes. These molecules are easily polarized to high levels using standard dynamic nuclear polarization (DNP) techniques and their 15N chemical shifts were found to be highly sensitive to pH. These probes displayed sharp 15N resonances and large differences in chemical shifts (Δδ >90 ppm) between their free base and protonated forms. These favorable features make these agents highly suitable candidates for the detection of small changes in tissue pH near physiological values.

Highly sensitive MR imaging agents that can accurately and rapidly monitor changes in pH would have diagnostic and prognostic value for many diseases. Here, we report an investigation of hyperpolarized 15 N-pyridine derivatives as ultrasensitive pH-sensitive imaging probes. These molecules are easily polarized to high levels using standard dynamic nuclear polarization (DNP) techniques and their 15 N chemical shifts were found to be highly sensitive to pH. These probes displayed sharp 15 N resonances and large differences in chemical shifts (Dd .90 ppm) between their free base and protonated forms. These favorable features make these agents highly suitable candidates for the detection of small changes in tissue pH near physiological values.
M R imaging agents with excellent pH-sensing capability and imaging sensitivity could become very useful tools for monitoring tissue acidosis that presents in many disease states including rapidly growing tumors [1][2][3] . Such pH-sensitive agents must have pK a values near the pH of interest, ideally around ,6.7, display large pH-induced changes in chemical shift, and have a suitable MR sensitivity for imaging pH in vivo. Several pH-sensitive probes reported previously rely on magnetic resonance spectroscopy (MRS) for readout of pH 4 . These include 1 H-5 , 19 F- 6 , and 31 P-based agents 7,8 . Chelated Gd(III) and Eu(III) complexes have also been developed as MRI agents for pH mapping based on pH-dependent relaxivity and chemical exchange saturation transfer (CEST) imaging 9,10 , respectively. Despite some success using the Gd(III)-based agents to image pH in vivo, these agents require a separate measure of agent concentration for pH calibration. CEST agents offer the possibility of obtaining a direct readout of pH using ratiometric methods but the image sensitivity of such agents remains problematical. Recently, it has been reported that the sensitivity of NMR insensitive nuclei can be significantly improved, .10,000-fold or more, by use of dynamic nuclear polarization (DNP) methods 11 . In DNP, the extremely high thermal polarization of free radical electrons is transferred to the nuclear spins of NMR-active nuclei, such as 13 C and 15 N, by microwave irradiation of a frozen sample. The transfer generates a non-equilibrium ''hyperpolarized'' (HP) spin state with dramatically improved NMR sensitivity that returns to thermal equilibrium as a function of spin-lattice relaxation time, T 1 . DNP has played a key role in following realtime metabolism of HP 13 C-labeled substrates such as [1-13 C]pyruvate in tumors, heart, and liver [12][13][14][15][16] . Hyperpolarized chemical probes that can in real-time monitor the pH, H 2 O 2 and redox state have also been investigated [17][18][19]  ) can be used to image pH of tumors in mice 15,20 . The same principle was later applied to measure intracellular pH (pHi) in isolated perfused hearts, based on intracellular generation of HP H 13 CO 3 2 / 13 CO 2 by oxidation of HP [1-13 C]pyruvate 21 . It is important to point out that the pH estimated using HP bicarbonate likely reflects a combination of intracellular (pHi) and extracellular pH (pHe) because CO 2 can freely diffuse through cell membranes 20 . The short T 1 of H 13 CO 3 2 is also a potential drawback for clinical translation of exogenous HP H 13 CO 3 2 . In another example, Jindal et al reported HP 89 Y-complexes as potential agents for chemical shift imaging (CSI) of pH by MRI 22 . Potential drawbacks for these complexes include the low c of 89 Y (low imaging sensitivity) and the relatively small pH-dependent chemical shifts (Dd,10 ppm from pH 5 to 9) observed for such systems.
Herein, we demonstrate the potential utility of using HP 15 N-pyridine derivatives as ultrasensitive pH imaging agents (Fig. 1). We first investigated the 15 N NMR signal enhancement by DNP of the sp 2 -hybridized nitrogens in pyridine derivatives (i.e. pyridine, 2,6-lutidine, 2-picoline, nicotinamide, and 2,4,6-collidine) and the 15 N chemical shift pH dependence. Previously, it has been shown that the 15 N chemical shifts of certain nitrogen functional groups were highly sensitive to protonation and a sharp chemical shift change near the pKa was observed 23 11,24 . It has also been shown that some 15 N-labeled functional groups such as 15 N-enriched choline (,4 min) 24 , and 15 N-enriched nitro compounds (,100 s) 25 have much longer T 1 than carbonyl 13 C centers.
Results and Discussion 15 N-NMR signal enhancement and spin-lattice relaxation time of pH probes. In this study, we aim to develop HP spectroscopic imaging agents that are highly sensitive to pH changes. Pyridine derivatives were chosen for evaluation because it is known that their 15 N resonances have relatively long T 1 's and are highly pH-sensitive 23 . The pKa values of pyridine derivatives are optimal for tissue pH measurements and can also be altered considerably by adding substituents. Therefore, it is highly possible that pyridine derivatives having suitable pKa values for any specific pH measurement applications can be designed and synthesized. Despite the excellent sensing capability, the role of 15 N NMR in molecular imaging is very limited mainly due to its poor sensitivity. To overcome such shortcoming, we investigated DNP hyperpolarization as a means to improve the 15 N NMR sensitivity of pH-sensitive pyridine derivatives. Our results showed that these pyridine derivatives can be well polarized by DNP with high 15 N signal enhancements. After dissolution into water, the 15 N signal enhancements of 5437 6 200, 8702 6 700, 7877 6 400, 10065 6 600, and 3598 6 700 were observed for 15 Npyridine, 15 N-2,6-lutidine, 15 N-2-picoline, 15 N-2,4,6-collidine and 15 N-nicotinamide, respectively. This level of enhancement is comparable to previously reported values for aromatic 15 N compounds 25 . The observed higher polarization of methyl-substituted pyridine compounds was likely due to their greater tendency to form frozen glasses when mixed with the DMSO/Sulfolane glassing agent 25 . 15 N NMR spectra of hyperpolarized and thermal samples of 15 N-pyridine are compared in Fig. 2a. Here, the 15 N NMR spectra of the HP sample was collected from a single 10-degree pulse while the thermally polarized spectra represent a total signal from 256 scans using 90degree pulses. It is important to note that both the HP and thermally polarized spectra were acquired from the same sample. The big difference in signal intensity demonstrates the tremendous signal enhancement achieved by DNP hyperpolarization. Fig. 2b shows an array of representative 15 N NMR spectra of HP 15 N-pyridine collected every 5 s using a 5-degree pulse after dissolution with water. By fitting such time-dependent 15 N signal intensity decay curves (Fig. 2c), the T 1 of HP 15 N-pyridine (pH 8.4), 15 N-2,6lutidine (pH 9.5), 15 N-2-picoline (pH 8.5), and 15 N-nicotinamide (pH 5.9) were found to be 41 6 3 s, 31 6 2 s, 38 6 2 s and 22 6 0.3 s, respectively (Supplementary Fig. S1 online). Due to poor water solubility, dissolution of 2,4,6-collidine required the use of methanol. Here, the measured T 1 was 36 6 4 s. The presence of methyl substituents on the pyridine ring results in a larger number of protons in close proximity to the nitrogen which results in more efficient dipoledipole relaxation of the pyridyl 15 N. Nevertheless, these T 1 values are comparable to many 13 C carboxyl groups at this same field currently used for metabolic imaging. The hyperpolarized 15 N signal of pyridinyl derivatives decayed more rapidly near physiological pH (Fig. 3a), likely reflecting an added relaxation pathway by the exchanging protons as these molecules become partially protonated 26 . As expected, the T 1 of 15 N-pyridine measured in plasma at 37uC was shorter (,11 s, Fig. 3b). While undesirable, this should not prevent the in vivo application of 15 N-labeled pyridine derivatives as pH probes because protonation equilibria are established nearly instantaneously. In comparison, the 13 C bicarbonate/carbon dioxide equilibrium system has been used successfully for imaging tissue pH in vivo even though the T 1 of the 13 C atoms in these molecules were only ,10 s 20 .
pH sensitivity of HP 15 N-pyridine derivatives. 15 N NMR has long been known to have a great sensing capability for different chemical environments such as acidity 23 and metal ion concentrations 27 . Protonation or metal chelation of some nitrogen-containing compounds can lead to a significant change in 15 N chemical shift, making them suitable MR sensors for pH or metal ions. For pyridine derivatives, the electronic properties of the sp 2 -hybridized, aromatic nitrogen center are greatly altered upon protonation leading to a significant change in  (Fig. 4b). The 15 N NMR linewidth of each probe was narrow in both the fully protonated or fully deprotonated forms but broaden somewhat at pH values near their respective pK a 's. This broadening reflects intermediate rates of exchange of protons between the 15 N atom and water 26 . The pK a values estimated by fitting the pH titration curves were 5.17 6 0.07 for pyridine, 6.60 6 0.02 for 2,6lutidine, 6.02 6 0.05 for 2-picoline, 7.65 6 0.05 for 2,4,6-collidine, and 4.14 6 0.02 for nicotinamide. These pKa values agreed well with values previously reported 28 . The 15 N chemical shift differences between the free base and fully protonated forms were all in the range, 88-94 ppm ( Table 1). The chemical shifts of all five 15 N enriched compounds changed linearly with pH at least over 61 pH units of each ligand pK a . The data demonstrate that these 15 N probes are quite sensitive to changes in pH (,60 ppm/pH unit) and that structures can be modified to fine-tune the probe to readout any desired pH value from 5 to 8.5.
To test the accuracy of these pH probes, a series of samples containing HP 15 N-pyridine or 15 N-2,6-lutidine with variable amounts of HCl added to the dissolution solution were examined by 15 N-NMR ( Supplementary Fig. S2 online). The two agents were chosen because of their long T 1 values. For each pH probe, the test was carried out over a pH range near the respective pKa values where the 15 N chemical shifts are extremely sensitive to pH. The pH of each sample was estimated from the observed 15 N chemical shifts (Fig. 4b) and also measured using a pH electrode 29 . Fig. 4c shows plots of pH as measured by 15 N-NMR versus a pH electrode. The results show an excellent correlation between the two measurements for both probes (R 2 . 0.99 for 2,6-lutidine and R 2 . 0.95 for pyridine), demonstrating the high accuracy of pH measurements by 15 N NMR of HP-pyridine derivatives.
Magnetic resonance spectroscopic imaging of HP 15 N-Pyridine. Chemical shift images of phantoms containing HP 15 N-enriched pyridine were collected to test the applicability of applying HP 15 Npyridine derivatives to distinguish pH differences. In this experiment, a solution of HP 15 N-pyridine was simultaneously injected into two NMR tubes inserted within a large NMR tube (see Supplementary  Fig. S3 online for the phantom setup). One of the small tubes contained a predetermined amount of sulfuric acid while the other contained sodium hydroxide. The imaging plane was positioned axially covering both tubes of HP-pyridine solutions (see 1 H image in Fig. 5). 15 N CSI data demonstrate that the 15 N signals of both basic and acidic HP-pyridine compartments were detectable by MRI. 15 N NMR spectra of selected voxels show 15 N resonances at 297.4 and 207.5 ppm reflecting the free base and protonated pyridine samples, respectively. CSI images of both basic and acidic pyridine displayed good localization of these two signals within the tubes (see CSI and merged image). The results show that magnetic resonance spectroscopic imaging (MRSI) of HP 15 N-enriched pyridine was able to distinguish different pH environments in adjacent spatial locations in a phantom. It is worth noting that the imaging of HP 15 N-pyridine was very rapid and a similar image of thermally polarized 15 N-enriched pyridine would have required a much higher 15 N spin concentration and a longer imaging time. These results strongly emphasize the sensitivity advantage of HP 15 N for molecular imaging applications over thermally polarized imaging practices. To the best of our  knowledge, these results demonstrate for the first time the feasibility of 15 N spectroscopic imaging of pH-sensitive agents, strongly emphasizing the sensitivity advantage of HP 15 N for molecular imaging applications.
Potential in vivo imaging applications of these agents include, but not limited to, pH imaging or MR spectroscopic assessment of tissue acidosis that is present in many diseases. However, many considerations must be taken into account before HP pyridine derivatives can be translated for in vivo evaluations. First, the HP pyridine agents should preferably be present as a free base or be partially protonated at the physiological pH. The long T 1 of the free base will allow for the HP signal to be retained much longer during the pre-injection quality controls of the agents. Additionally, the pH regions of the targeted tissues should not be very close to the pKa of the agents in order to avoid the rapid signal loss from fast water proton exchanges. Therefore, the ideal HP pyridine derivatives should have a pKa that is slightly below the pH of acidic tissues to be assessed in order to provide a relatively large chemical shift window and relatively long HP signal lifetime. The toxicity of these agents is also an important aspect that needs to be considered. Although the toxicity of pyridine is a concern, there are several naturally occurring pyridine derivatives that can be considered. Some of these compounds are present in the biological systems and play key roles in human physiology. For example, nicotinamide is a building block for nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) while nicotinic acid (vitamin B 3 ) and pyridoxyl derivatives (vitamin B 6 ) are essential human nutrients. Another derivative, picolinic acid, is a product of the kynurenine pathway and is a neuroprotective compound 30 . Chemical modifications of these molecules will be required for potential in vivo pH imaging applications to alter their pKa values toward the desirable range while preserving their biocompatibility. Future work will be focused on biocompatible pyridinyl compounds with favorable pK a values for pH imaging of tissue acidosis.

Conclusion
We have demonstrated the potential of using HP 15 N-pyridine derivatives as pH-sensitive probes for MRI. These molecules display large changes in 15 N chemical shift with pH and have very sharp chemical shift versus pH titration curves. The combination of hyperpolarization and pH-sensitive 15 N agents offers new opportunities to develop highly sensitive imaging agents with great sensing capability for the characterization of important biomarkers such as tissue acidity. Future work will be focused on in vivo pH imaging of acidic tumors. according to the manufacturer's procedures. The polarization was carried out at ,1.05 K with 94.055 GHz microwave irradiation for 2 h. The dissolution liquid of HP agents in distilled water (4 mL) from the polarizer was rapidly mixed with a predetermined volume of hydrochloric acid or sodium hydroxide in a 10-mm NMR tube to achieve the desired pH. 15 N-NMR spectra were acquired at room temperature (,23 uC) on a 400-MHz spectrometer using a 5 degree flip angle with a repetition time (TR) of 5 s. All 15 N peaks were externally referenced with 15 N-nitrobenzene (372 ppm). 15 N MRI were acquired on a 9.4 T Agilent vertical bore microimager (Agilent, USA). MR images and spectra were processed using ImageJ (NIH, USA) and ACD/SpecManager (ACD Labs, Canada). The T 1 , signal enhancement and pH titration experiments were investigated with the natural abundant 15 N compounds. 15 N-labeled pyridine was used in T 1 measurement in plasma and CSI experiments.

Methods
T 1 relaxation times and signal enhancements of HP pyridine derivatives. In a typical protocol, a dissolution liquid (4 mL) of HP-pyridine derivative was rapidly transferred into a 10-mm NMR tube. 15 N NMR acquisition was initiated once the transfer was complete. An array of 15 N spectra with one spectra recorded every 5 s was obtained (TR 5 5 s, flip angle 5 5 degree). 15   20-mM solution of a pyridine compound was used. 256 scans of 15 N NMR spectra were acquired using 90-degree pulses with a 500second delay (.5 times T 1 ). Enhancement levels were calculated as ratios of the 15 N signal from the two polarized states, taking into account for the different 15 N concentrations and number of scans (1 vs 256). PBS buffer solution (pH 5 7.4) was used as a dissolution solvent for T 1 measurements at the physiological pH. The final concentration of HP 15 N-pyridine or 2,6-lutidine was 2 mM. The plasma used in T 1 of HP 15 N-pyridine was obtained by centrifugation of whole rat blood to remove red blood cells. In this experiment, the dissolution liquid of HP 15 N-pyridine (2 mL) was well mixed with rat plasma (2 mL) in a 10-mm NMR tube. The final concentration of HP 15 N-pyridine was 13 mM. 15 N-NMR spectrum was acquired at ,37uC. Chemical shift imaging (CSI) of HP 15 N-pyridine. The phantom for this experiment was a 25-mm NMR tube encasing two 10-mm NMR tubes, with one pre-added sulfuric acid (200 mL, 7 M) and another had sodium hydroxide (200 mL, 5 M). The phantom was inserted into a 25-mm NMR tube containing DI water (,10 mL). The imaging plane was positioned axially across the phantom ( Supplementary Fig. S3a  online). 15 N CSI was acquired after the transfer was completed with a 10-s delay to allow for complete mixing. pH values of the HP-pyridine solutions measured by pH electrode in the small tubes were 2 and 11. 15   T 1 was calculated using Equation 1; [b] enhancements were measured after dissolution; [c] pKa was estimated by fitting pH titration curves. [d] T 1 of 2,4,6-collidine was estimated in methanol.